Effects of stable suppression of Group VIA phospholipase A2 expression on phospholipid content and composition, insulin secretion, and proliferation of INS-1 insulinoma cells

Studies involving pharmacologic inhibition or transient reduction of Group VIA phospholipase A2 (iPLA2beta) expression have suggested that it is a housekeeping enzyme that regulates cell 2-lysophosphatidylcholine (LPC) levels, rates of arachidonate incorporation into phospholipids, and degradation of excess phosphatidylcholine (PC). In insulin-secreting islet beta-cells and some other cells, in contrast, iPLA2beta signaling functions have been proposed. Using retroviral vectors, we prepared clonal INS-1 beta-cell lines in which iPLA2beta expression is stably suppressed by small interfering RNA. Two such iPLA2beta knockdown (iPLA2beta-KD) cell lines express less than 20% of the iPLA2beta of control INS-1 cell lines. The iPLA2beta-KD INS-1 cells exhibit impaired insulin secretory responses and reduced proliferation rates. Electrospray ionization mass spectrometric analyses of PC and LPC species that accumulate in INS-1 cells cultured with arachidonic acid suggest that 18:0/20:4-glycerophosphocholine (GPC) synthesis involves sn-2 remodeling to yield 16:0/20:4-GPC and then sn-1 remodeling via a 1-lyso/20:4-GPC intermediate. Electrospray ionization mass spectrometric analyses also indicate that the PC and LPC content and composition of iPLA2beta-KD and control INS-1 cells are nearly identical, as are the rates of arachidonate incorporation into PC and the composition and remodeling of other phospholipid classes. These findings indicate that iPLA2beta plays signaling or effector roles in beta-cell secretion and proliferation but that stable suppression of its expression does not affect beta-cell GPC lipid content or composition even under conditions in which LPC is being actively consumed by conversion to PC. This calls into question the generality of proposed housekeeping functions for iPLA2beta in PC homeostasis and remodeling.     

Studies of the role of group VI phospholipase A2 in fatty acid incorporation, phospholipid remodeling, lysophosphatidylcholine generation, and secretagogue-induced arachidonic acid release in pancreatic islets and insulinoma cells.

An 84-kDa group VI phospholipase A2 (iPLA2) that does not require Ca2+ for catalysis has been cloned from Chinese hamster ovary cells, murine P388D1 cells, and pancreatic islet beta-cells. A housekeeping role for iPLA2 in generating lysophosphatidylcholine (LPC) acceptors for arachidonic acid incorporation into phosphatidylcholine (PC) has been proposed because iPLA2 inhibition reduces LPC levels and suppresses arachidonate incorporation and phospholipid remodeling in P388D1 cells. Because islet beta-cell phospholipids are enriched in arachidonate, we have examined the role of iPLA2 in arachidonate incorporation into islets and INS-1 insulinoma cells. Inhibition of iPLA2 with a bromoenol lactone (BEL) suicide substrate did not suppress and generally enhanced [3H]arachidonate incorporation into these cells in the presence or absence of extracellular calcium at varied time points and BEL concentrations. Arachidonate incorporation into islet phospholipids involved deacylation-reacylation and not de novo synthesis, as indicated by experiments with varied extracellular glucose concentrations and by examining [14C]glucose incorporation into phospholipids. BEL also inhibited islet cytosolic phosphatidate phosphohydrolase (PAPH), but the PAPH inhibitor propranolol did not affect arachidonate incorporation into islet or INS-1 cell phospholipids. Inhibition of islet iPLA2 did not alter the phospholipid head-group classes into which [3H]arachidonate was initially incorporated or its subsequent transfer from PC to other lipids. Electrospray ionization mass spectrometric measurements indicated that inhibition of INS-1 cell iPLA2 accelerated arachidonate incorporation into PC and that inhibition of islet iPLA2 reduced LPC levels by 25%, suggesting that LPC mass does not limit arachidonate incorporation into islet PC. Gas chromatography/mass spectrometry measurements indicated that BEL but not propranolol suppressed insulin secretagogue-induced hydrolysis of arachidonate from islet phospholipids. In islets and INS-1 cells, iPLA2 is thus not required for arachidonate incorporation or phospholipid remodeling and may play other roles in these cells.     

Group VIB Ca2+-independent phospholipase A2gamma promotes cellular membrane hydrolysis and prostaglandin production in a manner distinct from other intracellular phospholipases A2

Although group VIA Ca2+-independent phospholipase A2beta (iPLA2beta) has been implicated in various cellular events, the functions of other iPLA2 isozymes remain largely elusive. In this study, we examined the cellular functions of group VIB iPLA2gamma. Lentiviral transfection of iPLA2gamma into HEK293 cells resulted in marked increases in spontaneous, stimulus-coupled, and cell death-associated release of arachidonic acid (AA), which was converted to prostaglandin E2 with preferred cyclooxygenase (COX)-1 coupling. Conversely, treatment of HEK293 cells with iPLA2gamma small interfering RNA significantly reduced AA release, indicating the participation of endogenous iPLA2gamma. iPLA2gamma protein appeared in multiple sizes according to cell types, and a 63-kDa form was localized mainly in peroxisomes. Electrospray ionization mass spectrometry of cellular phospholipids revealed that iPLA2gamma and other intracellular PLA2 enzymes acted on different phospholipid subclasses. Transfection of iPLA2gamma into HCA-7 cells also led to increased AA release and prostaglandin E2 synthesis via both COX-1 and COX-2, with a concomitant increase in cell growth. Immunohistochemistry of human colorectal cancer tissues showed elevated expression of iPLA2gamma in adenocarcinoma cells. These results collectively suggest distinct roles for iPLA2beta and iPLA2gamma in cellular homeostasis and signaling, a functional link between peroxisomal AA release and eicosanoid generation, and a potential contribution of iPLA2gamma to tumorigenesis.     

Tryptase activates calcium-independent phospholipase A2 and releases PGE2 in airway epithelial cells

Human small airway epithelial cells (HSAEC) form the boundary between the external environmental allergens and the internal lung milieu. Mast cells are present in human lung tissue interspersed within the pulmonary epithelium and can secrete a host of pre- and newly formed mediators from their granules, which may propagate small airway inflammation. In this study, tryptase stimulation of HSAEC increased membrane-associated, calcium-independent phospholipase A(2)gamma (iPLA(2)gamma) activity, resulting in increased arachidonic acid and PGE(2) release. These responses were inhibited by pretreating HSAEC with the iPLA(2)-selective inhibitor bromoenol lactone. The tryptase-stimulated PGE(2) production was inhibited by treating HSAEC with the cyclooxygenase (COX)-1-selective inhibitor SC-560 and the nonselective COX inhibitor aspirin but not by the COX-2-selective inhibitor CAY10404, indicating that the early release of arachidonic acid is metabolized by constitutive COX-1 to form PGE(2) in tryptase-stimulated HSAEC. Additionally, platelet-activating factor production and neutrophil adherence to tryptase-stimulated HSAEC was also increased. This complex response can set up a cascade of inflammatory mediator production in small airways. We speculate that selective inhibition of iPLA(2)gamma-mediated phospholipid hydrolysis may prove beneficial in inflammatory airway diseases.     

Substrate specificity of a CoA-dependent stearoyl transacylase from bovine testis membranes.

We identified a CoA-dependent stearoyl transacylase activity in bovine testis membranes, then examined the enzyme's specificity in mixed micelle systems containing the neutral detergent Triton X-100. The enzyme transferred stearoyl groups from a variety of phospholipids to sn-2-arachidonoyl lysophosphatidic acid (lysoPA), but showed very little palmitoyl transacylase activity. Its ability to transfer stearoyl groups was both donor- and acceptor-dependent. For example, it used weakly acidic phospholipids, such as sn-1-stearoyl-2-acyl species of phosphatidylinositol (PI), as donors, but did not use phosphatidylinositol-4,5-bisphosphate or sn-1-stearoyl-2-arachidonoyl phosphatidylcholine. Moreover, it used sn-2-acyl species of lysoPA and sn-2-arachidonoyl lysoPI as acceptors but did not use sn-2-arachidonoyl species of lysophosphatidylserine, lysophosphatidylethanolamine, or lysophosphatidylcholine. When taken together, our results raise the possibility that sn-1-stearoyl-2-acyl species of PI may be the primary acyl donors in the transacylase reaction in vivo, while sn-2-acyl species of lysoPA may be the primary acyl acceptors. Available evidence suggests that the PA that is formed may subsequently be converted into PI, but the metabolic fate of the other reaction product, sn-2-acyl lysoPI, remains to be determined.     

Studies of insulin secretory responses and of arachidonic acid incorporation into phospholipids of stably transfected insulinoma cells that overexpress group VIA phospholipase A2 (iPLA2beta ) indicate a signaling rather than a housekeeping role for iPLA2beta

A cytosolic 84-kDa group VIA phospholipase A(2) (iPLA(2)beta) that does not require Ca(2+) for catalysis has been cloned from several sources, including rat and human pancreatic islet beta-cells and murine P388D1 cells. Many potential iPLA(2)beta functions have been proposed, including a signaling role in beta-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA(2)beta function rest in part on effects of inhibiting iPLA(2)beta activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A(2). Manipulation of iPLA(2)beta expression by molecular biologic means is an alternative approach to study iPLA(2)beta functions, and we have used a retroviral construct containing iPLA(2)beta cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA(2)beta. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA(2)beta-overexpressing lines exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents, and BEL substantially attenuates stimulated secretion. Electrospray ionization mass spectrometric analyses of arachidonic acid incorporation into INS-1 cell PC indicate that neither overexpression nor inhibition of iPLA(2)beta affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA(2)beta indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA(2)beta associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA(2)beta in insulin-secreting beta-cells.     

Swiss 3T3 cells preferentially incorporate sn-2-arachidonoyl monoacylglycerol into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.

The sn-1-stearoyl-2-arachidonoyl phospholipids of animal cells appear to be formed by special mechanisms. To determine whether monoacylglycerol (MG) incorporation pathways are involved we incubated quiescent Swiss 3T3 cells with [3H]glycerol-labeled sn-2-arachidonoyl MG, then analyzed the radioactive cell lipids that accumulated. We also examined cell homogenates to identify enzyme activities that might promote the incorporation of sn-2-arachidonoyl MG into other cell lipids. The cell incubation experiments demonstrated rapid labeling of several lipids, including diacylglycerol, lysophosphatidic acid, phosphatidic acid, and phosphatidylinositol. They also demonstrated selective labeling of sn-1-stearoyl-2-arachidonoyl species of phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. The cell homogenate experiments identified an sn-2-acyl MG acyltransferase activity, an MG kinase activity that phosphorylates sn-2-arachidonoyl MG in preference to sn-2-oleoyl MG, and a stearoyl-specific acyl transferase activity that converts sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidic acid. The results also showed that this stearoyl transferase could act with other enzymes to convert sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol. The combined results indicate that Swiss 3T3 cells incorporate sn-2-arachidonoyl MG into phospholipids by at least two different pathways, including one that specifically forms sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.     

Role of calcium-independent phospholipases (iPLA(2)) in phosphatidylcholine metabolism

The proposed role of calcium-independent phospholipase A(2) (iPLA(2)) in membrane phospholipid homeostasis was tested by examining the perturbation of phosphatidylcholine metabolism by enzyme overexpression. There are alternatively spliced forms of murine iPLA(2) that were widely expressed in mouse tissues: a long form containing exon-9 that is membrane-associated and a short form lacking exon-9 that is distributed between the membrane and cytosolic fractions. Enforced expression of either iPLA(2) isoform led to a significant increase in intracellular free fatty acid, lysophosphatidylcholine, and GPC without a concomitant increase in the incorporation of either exogenous arachidonic acid or choline. The accumulation of lysophosphatidylcholine in iPLA(2)-expressing cells illustrates the limited capacity of cells for reacylation and degradation of lysophospholipids. Since iPLA(2) overexpression did not accelerate either phospholipid remodeling or phosphatidylcholine synthesis, this enzyme does play a determinant (rate-controlling?) role in either of these cellular processes.     

Activation of mitochondrial calcium-independent phospholipase A2γ (iPLA2γ) by divalent cations mediating arachidonate release and production of downstream eicosanoids

Calcium-independent phospholipase A(2)γ (iPLA(2)γ) (PNPLA8) is the predominant phospholipase activity in mammalian mitochondria. However, the chemical mechanisms that regulate its activity are unknown. Here, we utilize iPLA(2)γ gain of function and loss of function genetic models to demonstrate the robust activation of iPLA(2)γ in murine myocardial mitochondria by Ca(2+) or Mg(2+) ions. Calcium ion stimulated the production of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) from 1-palmitoyl-2-[(14)C]arachidonoyl-sn-glycero-3-phosphocholine during incubations with wild-type heart mitochondrial homogenates. Furthermore, incubation of mitochondrial homogenates from transgenic myocardium expressing iPLA(2)γ resulted in 13- and 25-fold increases in the initial rate of radiolabeled 2-AA-LPC and arachidonic acid (AA) production, respectively, in the presence of calcium ion. Mass spectrometric analysis of the products of calcium-activated hydrolysis of endogenous mitochondrial phospholipids in transgenic iPLA(2)γ mitochondria revealed the robust production of AA, 2-AA-LPC, and 2-docosahexaenoyl-LPC that was over 10-fold greater than wild-type mitochondria. The mechanism-based inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) (iPLA(2)γ selective), but not its enantiomer, (S)-BEL (iPLA(2)β selective) or pyrrolidine (cytosolic PLA(2)α selective), markedly attenuated Ca(2+)-dependent fatty acid release and polyunsaturated LPC production. Moreover, Ca(2+)-induced iPLA(2)γ activation was accompanied by the production of downstream eicosanoid metabolites that were nearly completely ablated by (R)-BEL or by genetic ablation of iPLA(2)γ. Intriguingly, Ca(2+)-induced iPLA(2)γ activation was completely inhibited by long-chain acyl-CoA (IC(50) ～20 μm) as well as by a nonhydrolyzable acyl-CoA thioether analog. Collectively, these results demonstrate that mitochondrial iPLA(2)γ is activated by divalent cations and inhibited by acyl-CoA modulating the generation of biologically active metabolites that regulate mitochondrial bioenergetic and signaling functions.     

A method for distinguishing 1-acyl from 2-acyl lysophosphatidylcholines generated in biological systems

Phospholipases A(1) and A(2) frequently coexist in biological systems. Generation of lysophosphatidylcholine (LPC) in such systems cannot be assigned to any of these types of enzymes unless the position of the fatty acid in the lysocompound can be unambiguously determined. We here present a simple method to achieve this purpose. It is based on the initial chemical acylation of the isolated LPC with a labeled fatty acid, followed by the enzymatic analysis of the resulting phosphatidylcholine (PC), using snake or bee venom phospholipase A(2). Thus, if treatment of the PC with this enzyme releases a labeled free fatty acid, it is demonstrated that the initial LPC was acylated at position sn-1, whereas if the product of hydrolysis yields labeled LPC, then the initial LPC was acylated at position sn-2. This is the first method devised to determine the source of LPC in the presence of mixtures of phospholipases A(1) and A(2) in complex biological systems.     

Production of 1,2-didocosahexaenoyl phosphatidylcholine by bonito muscle lysophosphatidylcholine/transacylase

1,2-Didocosahexaenoyl phosphatidylcholine (PC), which has highly unsaturated fatty acid at both sn-1 and sn-2 positions of glycerol, is a characteristic molecular species of bonito muscle. To examine the involvement of a de novo route in its synthesis, the molecular species of phosphatidic acid (PA) were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex, a novel phosphate-capture molecule. However, 1,2-didocosahexaenoyl species could not be detected. Next, 1,2-didocosahexaenoyl PC synthesis by the cytosolic lysophosphatidylcholine (LPC)/transacylase was examined using endogenous LPC from bonito muscle, in which the 2-docosahexaenoyl species is abundant. The LPC/transacylase synthesized 1,2-didocosahexaenoyl PC as the most abundant molecular species. For further characterization, the LPC/transacylase was purified to homogeneity from the 100,000 x g supernatant of bonito muscle. The isolated LPC/transacylase is a labile glycoprotein with molecular mass of 52 kDa including a 5-kDa sugar moiety. The LPC/transacylase showed a PC synthesis (transacylase activity) below and above the critical micelle concentration of substrate LPC, and fatty acid release (lysophospholipase activity) was always smaller than the transacylase activity, even with a monomeric substrate. These results suggest that the LPC/transacylase is responsible for the synthesis of 1,2-didocosahexaenoyl PC.     

Metabolomic analysis and identification of a role for the orphan human cytochrome P450 2W1 in selective oxidation of lysophospholipids

Human cytochrome P450 (P450) 2W1 is still considered an "orphan" because its physiological function is not characterized. To identify its substrate specificity, the purified recombinant enzyme was incubated with colorectal cancer extracts for untargeted substrate searches using an LC/MS-based metabolomic and isotopic labeling approach. In addition to previously reported fatty acids, oleyl (18:1) lysophosphatidylcholine (LPC, lysolecithin) was identified as a substrate for P450 2W1. Other human P450 enzymes tested showed little activity with 18:1 LPC. In addition to the LPCs, P450 2W1 acted on a series of other lysophospholipids, including lysophosphatidylinositol, lysophosphatidylserine, lysophosphatidylglycerol, lysophosphatidylethanolamine, and lysophosphatidic acid but not diacylphospholipids. P450 2W1 utilized sn-1 18:1 LPC as a substrate much more efficiently than the sn-2 isomer; we conclude that the sn-1 isomers of lysophospholipids are preferred substrates. Chiral analysis was performed on the 18:1 epoxidation products and showed enantio-selectivity for formation of (9R,10S) over (9R,10S). The kinetics and position specificities of P450 2W1-catalyzed oxygenation of lysophospholipids (16:0 LPC and 18:1 LPC) and fatty acids (C16:0 and C18:1) were also determined. Epoxidation and hydroxylation of 18:1 LPC are considerably more efficient than for the C18:1 free fatty acid.     

Human calcium-independent phospholipase A2 mediates lymphocyte proliferation

Activation of lymphocytes induces blastogenesis and cell division which is accompanied by membrane lipid metabolism such as increased fatty acid turnover. To date little is known about the enzymatic mechanism(s) regulating this process. Release of fatty acids such as arachidonic acid requires sn-2-deacylation catalyzed by a class of enzymes known as phospholipases A(2) (PLA(2), EC ). Herein, we confirm that human peripheral blood B or T lymphocytes (PBL) do not possess measurable levels of 85-kDa PLA(2) as assessed by Western immunoblot. Low levels of 14-kDa PLA(2) protein and activity were detectable in the particulate fraction of PBL and Jurkat cells. Western immunoblot analysis indicates that PBLs possess the calcium-independent PLA(2) (iPLA(2)) protein. Calcium-independent sn-2-acylhydrolytic activity was measurable in PBL cytosols and could be inhibited by the selective iPLA(2) inhibitor bromoenol lactone. Mitogen activation of PBLs resulted in maintenance of activity levels which remained constant over 72 h suggesting an important role for iPLA(2) in this proliferative process. Indeed, evaluation of iPLA(2) activity in cell cycle-arrested Jurkat T cell fractions revealed the highest iPLA(2) levels occurring at the G(2)/M phase. Addition of the iPLA(2) inhibitors, bromoenol lactone, or arachidonyl trifluoromethyl ketone (AAOCF(3)), inhibited both mitogen-induced PBL as well as Jurkat T cell proliferation. Moreover, specific depletion of iPLA(2) protein by antisense treatment also resulted in marked suppression of cell division. Inhibition of Jurkat cell proliferation was not associated with arrest at a particular phase of the cell cycle nor was it associated with apoptosis as assessed by flow cytometry. These findings provide the first evidence that iPLA(2) plays a key role in the lymphocyte proliferative response.     

Bradykinin Stimulates Arachidonic Acid Release Through the Sequential Actions of an sn-1 Diacylglycerol Lipase and a Monoacylglycerol Lipase

In cultured dorsal root ganglion (DRG) neurons prelabeled with [3H]arachidonic acid [( 3H]AA), bradykinin (BK) stimulation resulted in increased levels of radioactive diacylglycerol, monoacylglycerol, and free AA. The transient increases in content of radioactive diacylglycerol and monoacylglycerol preceded the increase in level of free AA, suggesting the contribution of a diacylglycerol lipase pathway to AA release. An analysis of the molecular species of diacylglycerols in unstimulated cultures revealed the presence of two primary [3H]AA-containing species, 1-palmitoyl-2-arachidonoyl and 1-stearoyl-2-arachidonoyl diacylglycerol. BK stimulation resulted in a preferential increase in content of 1-stearoyl-2-arachidonoyl diacylglycerol. When DRG cultures were labeled with [3H]stearic acid, treatment with BK increased the amount of label in diacylglycerol and free stearic acid, but not in monoacylglycerol. This result suggested that AA release occurred through the successive actions of an sn-1 diacylglycerol lipase and monoacylglycerol lipase. Other data supporting a diacylglycerol lipase pathway was the significant inhibition of [3H]AA release and consequent accumulation of diacylglycerol by RG 80267, which preferentially inhibits diacylglycerol lipase. Analysis of the molecular species profiles of individual phospholipids in DRG neurons indicated that phosphoinositide hydrolysis may account for a significant portion of the rapid increase in content of 1-stearoyl-2-arachidonoyl diacylglycerol. We were unable to obtain evidence that the phospholipase A2 pathway makes a significant contribution to BK-stimulated AA release in DRG cultures. Under our assay conditions there were no BK-stimulated increases in levels of radioactive lysophosphatidylinositol, lysophosphatidylcholine, or lysophosphatidylethanolamine in cultures prelabeled with [3H]inositol, [3H]choline, or [3H]-ethanolamine, respectively.     

Lysophosphatidylcholine as a death effector in the lipoapoptosis of hepatocytes

The pathogenesis of nonalcoholic steatohepatitis (NASH) is unclear, despite epidemiological data implicating FFAs. We studied the pathogenesis of NASH using lipoapoptosis models. Palmitic acid (PA) induced classical apoptosis of hepatocytes. PA-induced lipoapoptosis was inhibited by acyl-CoA synthetase inhibitor but not by ceramide synthesis inhibitors, suggesting that conversion products other than ceramide are involved. Phospholipase A(2) (PLA(2)) inhibitors blocked PA-induced hepatocyte death, suggesting an important role for PLA(2) and its product lysophosphatidylcholine (LPC). Small interfering RNA for Ca(2+)-independent phospholipase A(2) (iPLA(2)) inhibited the lipoapoptosis of hepatocytes. PA increased LPC content, which was reversed by iPLA(2) inhibitors. Pertussis toxin or dominant-negative Galpha(i) mutant inhibited hepatocyte death by PA or LPC acting through G-protein-coupled receptor (GPCR)/Galpha(i). PA decreased cardiolipin content and induced mitochondrial potential loss and cytochrome c translocation. Oleic acid inhibited PA-induced hepatocyte death by diverting PA to triglyceride and decreasing LPC content, suggesting that FFAs lead to steatosis or lipoapoptosis according to the abundance of saturated/unsaturated FFAs. LPC administration induced hepatitis in vivo. LPC content was increased in the liver specimens from NASH patients. These results demonstrate that LPC is a death effector in the lipoapoptosis of hepatocytes and suggest potential therapeutic values of PLA(2) inhibitors or GPCR/Galpha(i) inhibitors in NASH.     

Identification of hepatic peroxisomal phospholipase A(2) and characterization of arachidonic acid-containing choline glycerophospholipids in hepatic peroxisomes

Recently, a sequence encoding a novel mammalian calcium-independent phospholipase A(2) (iPLA(2)gamma) was identified in the human genome and subsequently cloned and expressed in Sf9 insect cells. Unexpectedly, expression studies in recombinant systems demonstrated the usage of multiple translation initiation codons resulting in different polypeptides. Herein, we demonstrate that hepatic iPLA(2)gamma is localized to rat liver peroxisomes, possesses a molecular mass of 63 kDa and that peroxisomal membranes are highly enriched in arachidonic acid-containing phospholipids. Collectively, these results provide the first demonstration of iPLA(2)gamma in mammalian tissue and suggest the possibility that iPLA(2)gamma can contribute to lipid second messenger generation by hydrolysis of peroxisomal arachidonic acid-containing phospholipids.     

Identification of calcium-independent phospholipase A2 (iPLA2) beta,and not iPLA2gamma,as the mediator of arginine vasopressin-induced arachidonic acid release in A-10 smooth muscle cells. Enantioselective mechanism-based discrimination of mammalian iPLA2s

The agonist-stimulated release of arachidonic acid (AA) from cellular phospholipids in many cell types (e.g. myocytes, beta-cells, and neurons) has been demonstrated to be primarily mediated by calcium-independent phospholipases A(2) (iPLA(2)s) that are inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL). Recently, the family of mammalian iPLA(2)s has been extended to include iPLA(2)gamma, which previously could not be pharmacologically distinguished from iPLA(2)beta. To determine whether iPLA(2)beta or iPLA(2)gamma (or both) were the enzymes responsible for arginine vasopressin (AVP)-induced AA release from A-10 cells, it became necessary to inhibit selectively iPLA(2)beta and iPLA(2)gamma in intact cells. We hypothesized that the R- and S-enantiomers of BEL would possess different inhibitory potencies for iPLA(2)beta and iPLA(2)gamma. Accordingly, racemic BEL was separated into its enantiomeric constituents by chiral high pressure liquid chromatography. Remarkably, (S)-BEL was approximately an order of magnitude more selective for iPLA(2)beta in comparison to iPLA(2)gamma. Conversely, (R)-BEL was approximately an order of magnitude more selective for iPLA(2)gamma than iPLA(2)beta. The AVP-induced liberation of AA from A-10 cells was selectively inhibited by (S)-BEL (IC(50) approximately 2 microm) but not (R)-BEL, demonstrating that the overwhelming majority of AA release is because of iPLA(2)beta and not iPLA(2)gamma activity. Furthermore, pretreatment of A-10 cells with (S)-BEL did not prevent AVP-induced MAPK phosphorylation or protein kinase C translocation. Finally, two different cell-permeable protein kinase C activators (phorbol-12-myristate-13-acetate and 1,2-dioctanoyl-sn-glycerol) could not restore the ability of A-10 cells to release AA after exposure to (S)-BEL, thus supporting the downstream role of iPLA(2)beta in AVP-induced AA release.     

The signal molecule lysophosphatidylcholine in Eschscholzia californica is rapidly metabolized by reacylation

In cultured cells of California poppy (Eschscholzia californica), lysophosphatidylcholine (LPC) triggers a signal path that finally induces alkaloid biosynthesis. LPC is transiently generated by elicitor-activated phospholipase A(2) of the plasma membrane. Externally added LPC is rapidly acylated by a membrane-bound enzyme that shows the highest specific activity in the purified plasma membrane. The fatty acid incorporated into the sn-2 position of LPC is preferentially linoleic (18:2), which is the most abundant acyl component in the PC species of Eschscholzia cells, but a minor component of the pool of free fatty acids. The fatty acid at the sn-1 position of LPC is less important for substrate specificity. The capacity of LPC acylation by intact cells or isolated plasma membranes by far exceeds the rate of LPC generation by activated phospholipase A(2) and is not limited by the availability of acyl donors. Metabolites other than phosphatidylcholine (PC) were not significantly produced from labeled LPC within 20 min, indicating that lysophospholipases are not significantly contributing to the short-time metabolism of LPC. It is concluded that reacylation to PC is the dominating process in the detoxication of LPC and ensures the transient character of its steady state concentrations, even at maximum phospholipase A(2) activities.     

Involvement of calcium-independent phospholipase A2 in uterine stromal cell phospholipid remodelling.

The role of Ca2+-independent phospholipase A2 (iPLA2) in arachidonic (AA) and docosahexaenoic (DHA) acid incorporation and phospholipid remodelling in rat uterine stromal cells (UIII cells) was studied. Incorporation of AA and DHA into UIII cell phospholipids was Ca2+-independent. Bromoenollactone (BEL), a potent inhibitor of iPLA2, reduced lysophosphatidylcholine level and AA incorporation into phospholipids by approximately 20%. DHA incorporation was not affected by BEL, indicating that the pathways for AA and DHA incorporation are partially different. In control cells, the transfer of AA occurred mainly from diacyl-glycerophosphocholine (GroPCho) to alkenylacyl-glycerophosphoethanolamine (GroPEtn) and to a lesser extent from diacyl-GroPCho to diacyl-GroPEtn. [3H]DHA was redistributed from diacyl-GroPCho and alkylacyl-GroPEtn to alkenylacyl-GroPEtn. BEL treatment inhibited completely the redistributrion of AA within diacyl-GroPCho and diacyl -GroPEtn and reduced the [3H]DHA content of diacyl-GroPEtn, indicating that a BEL-sensitive iPLA2 controls the redistribution of polyunsaturated fatty acids to diacyl-GroPEtn. In contrast the redistribution of radioactive AA and DHA to alkenylacyl-GroPEtn was almost insensitive to BEL. The analysis of substrate specificity and BEL sensitivity of iPLA2 activity indicates that UIII cells exhibit at least two isoforms of iPLA2, one of which is BEL-sensitive and quite selective of diacyl species, and another one that is insensitive to BEL and selective for alkenylacyl-GroPEtn. Taken together, these results suggest that several iPLA2 participate independently in the remodelling of UIII cell phospholipids.